Liquid chromatography is a method by which various species from a complex mixture can be separated out into their individual components. The individual species or components will elute from the liquid chromatography system at substantially different times.
Known liquid chromatography systems include High Performance Liquid Chromatography (HPLC) systems incorporating a pumping system which comprises two solvent channels A,B. By convention solvent channel A comprises an aqueous solvent or solution (e.g. HPLC grade water with 0.1% acid) and solvent channel B comprises an organic solvent (e.g. acetonitrite or methanol with 0.1% acid). The aqueous solvent or solution A and the organic solvent B are mixed so as to provide an isocratic flow. A sample or analyte to be analysed is then introduced into the mixed solvent flow. The sample may be introduced into the mixed solvent flow either manually or by means of an auto-sampler.
The sample or analyte together with solvent mixture is then passed to an analytical column which is commonly filled with stationary phase (e.g. 5 μm silicon beads). Initially the composition of mixed solvent is set so as to comprise predominantly aqueous solvent or solution from solvent channel A. However, the proportion of organic solvent B to aqueous solvent or solution A is slowly increased in a linear manner over a period of time. Components in the liquid which are initially trapped on the analytical column will begin to become mobile again as the organic solvent gradient increases i.e. as the proportion of organic solvent B in the solvent mixture increases. For example, the relative ratio of the flow rate from the two solvent channels may be linearly varied so that, for example, the solvent mixture initially comprises ˜1% organic solvent but the concentration of the solvent mixture progressively increases until the solvent mixture comprises 60% organic solvent B after a period of time of e.g. 60 minutes. As the relative composition of the mixture of the two solvents A,B is varied, different species become released from the stationary phase of the column and are subsequently detected by various means at the output to the analytical column.
The inside or internal diameters of analytical columns used in liquid chromatography applications can vary quite considerably. For example, the inside or internal diameter of an analytical column may be less than 50 μm in some applications whereas in other applications the inside or internal diameter may be in excess of 4.6 mm. The delivery flow rate required from the pumping system increases as the inside or internal diameter of the analytical column is increased and the delivery flow rate may, for example, range from several nanolitres per minute to several millilitres per minute.
It is common to use a direct flow arrangement wherein the delivery flow is passed direct to the analytical column and then on to an analytical instrument (e.g. mass spectrometer) without splitting the flow. However, there are circumstances wherein a direct flow arrangement is unsuitable.
In order to provide an accurate gradient at low flow rates (e.g. a few nanolites per minute) it is often necessary to split the delivery flow from a liquid chromatograph before the analytical instrument. There are two relatively common situations where it may, for example, be necessary to split the delivery flow. The first situation is when a large diameter HPLC analytical column is used. Conventional standard large diameter HPLC analytical columns have an internal diameter of 4.6 mm. Columns having an internal diameter of 4.6 mm are an industry standard and are reliable and robust. Such columns can also handle large quantities which can be useful in purification processes such as fraction collection. However, such large diameter columns commonly require relatively high flow rates of several millilitres per minute. Whilst it is not problematic to provide such flow rates to the analytical column, flow rates of several millilitres per minute can be too high a flow rate to be handled directly by, for example, an Electrospray Ionisation ion source which may be arranged to receive and ionise the flow eluting from the column. Relatively high flow rates may be particularly unsuitable for an Electrospray Ionisation ion source especially if the solvent mixture being used to push the sample through the analytical column contains a relatively high percentage or proportion of water. Accordingly, it then becomes necessary to split the flow either downstream from or upstream of the HPLC column so that only a proportion of the flow then passes directly to the Electrospray Ionisation ion source. The rest of the flow may be either simply dumped to waste or alternatively a specific component of interest may be collected in a vial in a process known as fraction collection.
The second situation where it may be necessary to the split the delivery flow is when using a nano-flow HPLC system. Nano-flow HPLC systems commonly utilise small internal diameter columns typically having an internal diameter <360 μm. Nano-flow HPLC systems therefore by their nature operate at relatively low flow rates typically in the range of 100 nl/min to 1000 nl/min i.e. flow rates 3-4 orders of magnitude lower than typical flow rates used with 4.6 mm internal diameter columns. A column having a small internal diameter may be used, for example, when only a very small amount of sample is available. For example, a nano-flow HPLC system may be used when analysing samples of less than 100 femto-mole of a protein digest extracted from human cells. However, since HPLC pumps are relatively poor at providing an accurate, stable and reproducible solvent gradient at such relatively low flow rates, it is known to run the pumps from solvent channels A,B at relatively higher flow rates but then to split the delivery flow before the nano-flow column so that only a much lower flow rate of delivery fluid passes through and on to the nano-flow HPLC column.
Electrospray Ionisation is a commonly used technique in mass spectrometry wherein species present in a flowing solution are ionised by the application of a high voltage to an electrospray probe. Electrospray ionisation is sometimes referred to as being a soft ionisation technique since the resulting ions produced by the ion source typically comprise relatively large molecular weight species (e.g. peptides) which can then be detected as intact ions by a mass analyser. Electrospray ionisation can be achieved at several different flow rates ranging from several nl/min (i.e. nano-flow rates) to flow rates of several ml/min.
The ion counts observed in a mass spectrometer during Electrospray ionisation are not, to a first approximation at least, flow rate dependent and therefore large sensitivity gains for the same signal to noise ratios can be achieved at lower flow rates due to much lower sample consumption.
A liquid chromatography system used in conjunction with an Electrospray Ionisation ion source mass spectrometer (LCMS) or a tandem mass spectrometer (LCMS/MS) represents a powerful analytical instrument which is widely used in many laboratories around the world. However, a limitation on the quality of data which can be achieved with low abundance species when using a liquid chromatography system coupled to a mass spectrometer is the relatively short time that any particular analyte species is actually present in the Electrospray Ionisation ion source. This also has the effect that the number of different MS/MS product ion mass spectra which can be performed and recorded is limited by the length of time that any species of parent ion is present within the ion source. This length of time is determined by the peak elution profile for the particular column being used.
It is known to attempt to effectively extend the time that a peak elutes by reducing the flow rate when species of interest are identified by a mass spectrometer. This technique is known as peak parking or variable flow chromatography. Reducing the flow rate in theory at least enables species of interest to be present in an ion source for longer periods of time without any loss of ion counts per scan.
U.S. Pat. No. 6,139,734 describes a method of variable flow chromatography wherein the flow rate is varied based upon the split ratio of different restrictors. The method described relies upon the use of two different delivery split flow ratios to determine a normal flow rate and a reduced flow rate. However, this approach suffers from the problem that the pressure equilibration is not instantaneous. Furthermore, the restrictors may become clogged causing differences in flow rate. A yet further problem with the disclosed variable flow approach is that with narrow peak elution times e.g. <0.5 min for a column having an internal diameter of 75 μm, the analyte corresponding to the eluting peak may have already completely passed through the ion source by the time that the reduced flow rate is actually fully established.
US 2002/0072126 describes another approach wherein a valve positioned post-column is switched and the species are eluted into the mass spectrometer at a low flow rate using a syringe pump. The post-column valve switches when species of interest are detected. The gradient delivery pump flow rate is halted and the column output blocked during a park event. A syringe pump then continues to elute the species into the ion source at a reduced flow rate. However, the use of a post-column valve leads to the introduction of a dead volume which is detrimental both to chromatographic performance and chromatographic resolution. The known method of using a post-column valve to enable variable flow chromatography is therefore particularly disadvantageous.
It is therefore desired to provide an improved liquid chromatography system which preferably does not suffer from some or all of the problems encountered with known liquid chromatography systems which employ variable flow rates.